Gene cloning and manipulation

6 important questions on Gene cloning and manipulation

How do we isolate and manipulate gene fragments?

Cutting, pasting, amplify in bacteria, gene manipulation or DNA sequencing

What are restrictions enzymes?

A protein isolated from bacteria that cleaves DNA sequences at sequence-specific sites
  • Type II: one enzymes recognizes and cuts the actual sequence and another enzyme methylates
    • recognize palindromes
    • works as dimers to recognize the sequence in both strands
    • create sticky or blunt ends when cutting DNA

What are the difference between sticky and blunt ends?

Sticky ends are when one strand is longer than the othe and with blunt ends  both strands are the same length.
  • the vector needs also te be sticky so it can bind to the plasmid very effective
  • blunt ends are not limited like sticky, but are ineffective
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What is pasting (ligating)?

Pasting is done after cutting, DNA ligase restore the phosphodiester bond by hydrolyzing of ATP

What are the specifications of a cloning vector?

  1. Origin of replication
  2. high copy number
  3. selection marker (antibiotic resistance gene)
    • They help in identifying transformants and selectively allowing their growth whereas eliminating non-transformants in cloning vector
  4. unique restriction sites to inset DNA
  5. simple method to establish recombinants (blue/white screening)

Why are vectors used with topoisomerase?

PCR fragments can be ligated directly into specific factors using TOPO

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